Successful tick-rickettsiae symbioses become stable as each organism's struggle for survival levels to a state of tolerance. Research demonstrates that Rickettsia montanensis elicits a defense response from the hard tick, Dermacentor variabilis. To establish a niche within a tick, rickettsiae must evade that response. Defining the biology that drives the transmission of vector-borne diseases has implications for public health. For instance, deciphering the underpinnings of the balance between tick defense and rickettsial invasion forms the basis of research that aims to interrupt transmission of vector-borne diseases. NIAID identified disruption of disease transmission by arthropods as a major research focus in its publication Planning for the 21st Century. My long-term goal is to understand how vector-borne pathogens colonize arthropods in the presence of an active host defense response. Our preliminary studies support our central hypothesis that D. variabilis-Kunitz-type protease inhibitor (Dv-KPI) limits rickettsial infection of the tick midgut by inactivating a rickettsial colonization factor. First, antibody-mediated neutralization of Dv-KPI results in increased rickettsial colonization in vitro. Second, RNAi-mediated knockdown of Dv-KPI in ticks results in an increase in R. montanensis burden in the midgut. Third, a bacterial affinity pulldown assay shows that Dv-KPI associates with R. montanensis. Fourth, a preliminary pulldown study suggests that Dv-KPI precipitates a rickettsial ligand. To address my long-term goal, NIAID's research focus and the central hypothesis, two aims are proposed. Specific Aim I - To determine if Dv-KPI limits R. montanensis entry and transmigration of the D. variabilis midgut. By suppressing Dv-KPI function in the tick, we will test further the effect that Dv-KPI has on rickettsial colonization and transmigration though the tick midgut. We will also investigate if entry is limited at the host cell perimeter or through endosomal escape. Specific Aim II - To identify the rickettsial ligand(s) for Dv-KPI. Using pulldown and Co-IP methods, we will identify and confirm the identity of the interacting rickettsial ligand(s). Completion of these aims will help to define the molecular attributes of the tick-rickettsiae interaction, which is central to the natural transmission cycle that leads to human infection. RELEVANCE (See instructions): Dv-KPI's observed affect on rickettsial growth forms the basis of our studies to investigate the factors that contribute to a state of tolerance between the tick and rickettsiae. Exploiting this knowledge to develop methods to interrupt tick transmission of pathogenic rickettsiae to humans addresses NIAID's research focus to disrupt disease transmission by arthropods.